tips for effective use of blast and other ncbi tools

Matthew McNeill, PhD

Tips for effective use of BLAST and other NCBI tools

8/23/20161

Introduction: What is NCBI?National Center for Biotechnology Information

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http://www.ncbi.nlm.nih.gov/

Introduction: NCBI’s available tools

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http://www.ncbi.nlm.nih.gov/home/analyze.shtml

Introduction: NCBI’s available tools

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http://www.ncbi.nlm.nih.gov/home/analyze.shtml

User story: Previously published paper

• A lncRNA regulates a network of genes involved in cancer processes

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6Sanchez et al., Nature Communications 5,Article number:5812

User story: We want to follow up on this work

Question: You have a collection of cancer cell lines. Does this lncRNA regulate the same network?

Selected tools:CRISPR – knockout lncRNA

qPCR – Analyze RNA expression of network

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User story: We want to follow up on this work

Question: You have a collection of cancer cell lines. Does this lncRNA regulate the same network?

Selected tools:CRISPR – knockout lncRNA

qPCR – Analyze RNA expression of network

Common theme when using genetic/ genomic tools: Was my assay specific?

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User story: Getting your gene sequences

• Identify your genes

• Downloading sequences

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User story: Getting your gene sequences



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User story: Gene list


lncRNA:

PR-‐lncRNA-‐1

Downstream genes:

TP53I3TGFB2SERPINB6POLA1PDK1LPPDPP4TNFRSF10DNCAPD3BCKDHBTRIO

User story: Identify your gene listTranslating IDs• Many options to consider

– Genome build– Gene Symbol/ Gene name– RefSeq Accession number.version

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User story: Identify your gene listTranslating IDs• Many options to consider

– Genome build– Gene Symbol/ Gene name– RefSeq Accession number.version

• Note:– NCBI is phasing out GI numbers– Read more here: https://www.ncbi.nlm.nih.gov/news/03-02-2016-phase-

out-of-GI-numbers/

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User story: Identify your gene listTranslating IDs—Genome build• Many options to consider

– Genome build• GRCh37/ hg19• GRCh38• GRCh38.p2

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User story: Identify your gene listTranslating IDs—annotations

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http://www.ncbi.nlm.nih.gov/

User story: Identify your gene listTranslating IDs—annotations—gene symbol

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http://www.ncbi.nlm.nih.gov/gene/?term=TP53I3

User story: Identify your gene listTranslating IDs—annotations—gene name

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User story: Identify your gene listTranslating IDs—annotations—gene alias

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User story: Identify your gene listTranslating IDs—annotations—RefSeq mRNA accession

20http://www.ncbi.nlm.nih.gov/gene/9540

User story: Identify your gene listTranslating IDs—annotations—RefSeq mRNA accession


NM_001206802

User story: Identify your gene listTranslating IDs—annotations—RefSeq mRNA accession.version


NM_001206802.2

User story: Identify your gene listTranslating IDs—annotations

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Gene symbol RefSeq mRNA accession

User story: Identify your gene listTranslating IDs—Annotations

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https://biodbnet-‐abcc.ncifcrf.gov/db/db2db.php



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Gene symbol RefSeq mRNA accessionNMXM

NR

User story: Getting your gene sequencesImportant background



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User story: Identify your gene listDownloading FASTA sequences


User story: Identify your gene listBatch Entrez

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http://www.ncbi.nlm.nih.gov/sites/batchentrez

User story: Identify your gene listBatch Entrez

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http://www.ncbi.nlm.nih.gov/sites/batchentrez

User story: Identify your gene listLog file page

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User story: Identify your gene listDownloading output

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User story: Identify your gene list FASTA file format>gi|332205880|ref|NM_001206802.2| Homo sapiens tumor protein p53 inducible protein 3 (TP53I3), transcript variant 3, mRNA ACAATATGTTAGCCGTGCACTTTGACAAGCCGGGAGGACCGGAAAACCTCTACGTGAAGGAGGTGGCCAA GCCGAGCCCGGGGGAGGGTGAAGTCCTCCTGAAGGTGGCGGCCAGCGCCCTGAACCGGGCGGACTTAATG CAGAGACAAGGCCAGTATGACCCACCTCCAGGAGCCAGCAACATTTTGGGACTTGAGGCATCTGGACATG TGGCAGAGCTGGGGCCTGGCTGCCAGGGACACTGGAAGATCGGGGACACAGCCATGGCTCTGCTCCCCGG TGGGGGCCAGGCTCAGTACGTCACTGTCCCCGAAGGGCTCCTCATGCCTATCCCAGAGGGATTGACCCTG ACCCAGGCTGCAGCCATCCCAGAGGCCTGGCTCACCGCCTTCCAGCTGTTACATCTTGTGGGAAATGTTC AGGCTGGAGACTATGTGCTAATCCATGCAGGACTGAGTGGTGTGGGCACAGCTGCTATCCAACTCACCCG GATGGCTGGAGCTATTCCTCTGGTCACAGCTGGCTCCCAGAAGAAGCTTCAAATGGCAGAAAAGCTTGGA GCAGCTGCTGGATTCAATTACAAAAAAGAGGATTTCTCTGAAGCAACGCTGAAATTCACCAAAGTACAAG CAAATGCTGGTGAATGCTTTCACGGAGCAAATTCTGCCTCACTTCTCCACGGAGGGCCCCCAACGTCTGC TGCCGGTTCTGGACAGAATCTACCCAGTGACCGAAATCCAGGAGGCCCATAAGTACATGGAGGCCAACAA GAACATAGGCAAGATCGTCCTGGAACTGCCCCAGTGAAGGAGGATGGGGCAGGACAGGACGCGGCCACCC CAGGCCTTTCCAGAGCAAACCTGGAGAAGATTCACAATAGACAGGCCAAGAAACCCGGTGCTTCCTCCAG AGCCGTTTAAAGCTGATATGAGGAAATAAAGAGTGAACTGGAAAAAAAAAA

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http://www.ncbi.nlm.nih.gov/nuccore/332205880?report=fasta


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5ʹ′

3ʹ′

User story: Getting your sequencesLearned so far• There are many identifiers that can be used for a gene, and those

identifiers are often updated. NCBI tracks update information.

• NCBI provides the sequence of genetic/ genomic elements for easy download individually or as batches.

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User story: Checking for off-target CRISPR eventsCRISPR—general overview

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https://www.idtdna.com/pages/products/genome-‐editing/crispr-‐cas9


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User story: Checking for off-target captureUsing BLAST• BLAST = Basic

Local Alignment Search Tool

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https://BLAST.ncbi.nlm.nih.gov/Blast.cgi

User story: Checking for off-target captureUsing BLAST• BLAST = Basic

Local Alignment Search Tool

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https://BLAST.ncbi.nlm.nih.gov/Blast.cgi

User story: Checking for off-target CRISPR eventsUsing BLASTN—optional parameters• Example guide RNA (crRNA) targeting PR-lncRNA-1: TTCCAAGTGGCTAAAACTAC(AGG)

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User story: Checking for off-target CRISPR eventsUsing BLASTN—optional parameters

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User story: Checking for off-target CRISPR eventsUsing BLASTN—output

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Perfect Match


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Off-‐Target Match


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Off-‐Target Match

User story: Checking for off-target CRISPR eventsLearned so far• Blast is a powerful tool to look for likely off-target CRISPR activity

• Correctly parsing your BLAST output improves off-target characterization

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User story: Checking off-target qPCR primersPCR—general overview

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Typicaldiagram


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Typicaldiagram

First cycle


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Typicaldiagram

First cycle

Second cycle

User story: Checking for off-target qPCR primersPrimer BLAST—overview

63https://BLAST.ncbi.nlm.nih.gov/Blast.cgi


64https://BLAST.ncbi.nlm.nih.gov/Blast.cgi


65https://www.ncbi.nlm.nih.gov/tools/primer-‐BLAST/index.cgi?LINK_LOC=BlastHome

User story: Checking for off-target qPCR primersPrimer BLAST—optional parameters

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User story: Checking for off-target qPCR primersPrimer BLAST—output

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User story: Analyze expression of your gene network

• Design qPCR primers

• Check primers for specificity, similar to lncRNA

• Order primers!

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User story: Checking for off-target qPCR primersLearned so far• PCR primers are consumed when they amplify a target.

• Off-target amplification will decrease the efficiency of on-target characterization for both SYBR and probe-based assays.

• Primer BLAST is a powerful tool to identify off-target regions that may be amplified.

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Summary: Covered tools

• Gene lookup—Gene database• Gene Symbol Translation—bioDB• Fasta Sequence Download—Gene database, Batch entrez• Single Sequence Uniqueness—BLASTN• Primer Uniqueness—Primer BLAST

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Conclusions

• NCBI provides a powerful suite of tools

• Checking for off-target hybridization, annealing, and amplification is important for genetic and genomic studies

• Proper use of settings for each informatics tools improves results

• For questions about anything we discussed, email: [email protected]

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Todd AdamsonNicola Brookman-AmissahSean McCallHans PackerMaureen Young

Thanks

Nick DowneyElisabeth Wagner

Aurita Menezes

Yu Wang

Available products

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Alt-R™ CRISPR-Cas9 System

• Cas9 protein, custom guide RNAs, and controls for genome editing• https://www.idtdna.com/pages/products/genome-editing/crispr-cas9

PrimeTime® qPCR Assays

• Predesigned primers, probes, multiple formats

• https://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers

tips for effective use of blast and other ncbi tools

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